http://informahealthcare.com/cts ISSN: 0300-8207 (print), 1607-8438 (electronic) Connect Tissue Res, 2014; 55(5–6): 357–366 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03008207.2014.951441
ORIGINAL RESEARCH
Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption Veronika Oralova´1,2, Ivana Chlasta´kova´3, Ralf Johannes Radlanski4, and Eva Matalova´1,3
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1
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Brno, Czech Republic, 2Department of Experimental Biology, Masaryk University, Brno, Czech Republic, 3Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic, and 4Department of Craniofacial Developmental Biology, Charite´-Universita¨tsmedizin, Berlin, Germany
Abstract
Keywords
Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth– bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.
Bone morphogenetic protein, bone resorption, bone apposition, periodontal ligament, three-dimensional images
Introduction The tooth is a typical example of an organ formed by epithelial–mesenchymal interactions (1). Different morphogens [bone morphogenetic protein (BMPs), WNTs, FGFs and HHs] participate in odontogenesis and related osteogenesis. Proper dentition is formed by functional reciprocal interplay of the tooth and surrounding structures (2,3). Tooth eruption is a highly regulated multifactorial process involving formation and reorganization of tissues coordinated by a complex set of molecular events. According to previous studies, biological tooth movement in mice is a result of osteoclastic bone resorption combined with alveolar bone and cementum formation (4,5). Prior to eruption (around postnatal stage 10), mononuclear cells move to the dental follicle and fuse to form osteoclasts, which resorb alveolar bone to create the eruption pathway (6). In addition, remodelling of the collagenous Correspondence: Ms Veronika Oralova´, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Veveri 97, Brno 602 00, Czech Republic. Tel: +420-532-290-163. Fax: +420-541212-988. E-mail: [email protected]
History Received 1 April 2014 Revised 23 July 2014 Accepted 26 July 2014 Published online 26 August 2014
extracellular matrix, periodontal ligaments (PDL) and alveolar bone was identified as a key molecular process facilitating tooth movement (7,8). Bone morphogenetic proteins (BMPs) are secretory signalling molecules belonging to the transforming growth factor-b (TGF-b) superfamily of polypeptides. Along with the capability to induce ectopic bone formation, they have other important signalling functions, such as governing cell proliferation, differentiation and apoptosis (9). BMPs were described to be expressed at different stages throughout tooth development and have central roles in tooth initiation and morphogenesis (10). BMP2 and BMP4 were shown to mimic some of the functions of the dental epithelium and induce tooth germ initiation in the mesenchyme (11). Moreover, BMP2 and BMP4 are also involved in osteoinduction (12,13). BMP2 and BMP7 stimulate mouse mesenchymal cells to differentiate into osteoblasts (14). BMP2 secreted in the basal region of the mouse dental follicle (DF) commits these cells towards the cementoblast/osteoblast phenotype (15) and supports the expression of cementum attachment protein (CA) in human periodontal ligament cells (16).
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BMP2, -3 and -7 were reported in the pre-dentine, dentine, osteoblasts, osteocytes, osteoid, chondrocytes, alveolar bone, cementum, PDL and other craniofacial structures (17,18). BMP6 was shown to function at early and late stages of osteogenic differentiation in human dental follicle cells (12). BMP6 was found to be upregulated in human PDL, suggesting the role of this protein in tooth eruption (19,20). BMP6 expression was found in cultured pulp cells and, together with other BMPs, BMP6 appears to be potent protein for future treatment strategies of bone regeneration (21). BMP6 knockout mice are viable, no dental defects have been reported, and the only skeletal alterations published so far are delayed ossification of the sternum (22) and decreased bone volume/ trabecular volume in the long bones (23). Knowledge about BMP6 in bones comes mostly from endochondral bones; nevertheless, local injection of a small-interfering RNA (siRNA) targeting BMP6 inhibited alveolar bone formation and subsequent tooth eruption, despite the fact that the eruption pathway was formed (20). The aim of this study was to further contribute to knowledge about BMP6 in relation to tooth eruption by analysis of its temporospatial expression pattern in mandibular molar teeth and surrounding structures, particularly the intramembranous bone, during pre-eruption and eruption stages of the first mouse molar tooth (M1). Attention was paid to localization of BMP6 and its correlation with proliferation, differentiation and apoptosis, as well as bone apposition/ resorption. Additionally, the pattern of BMP6 protein was matched with localization of other BMPs (BMP2 and BMP7). With respect to different bone environments during first/ second and third molar eruptions (24), distribution of BMP6 protein was compared in the bone around the first and third mouse molars. Moreover, an overview of tooth–bone development was documented using three-dimensional models (3D).
Materials and methods Animals Wild-type mice were obtained from the Breeding Unit of the Masaryk University in Brno. Heads of pups corresponding to postnatal (P) days up to the P26 were collected for sampling. All procedures were carried out according to the experimental protocol approved by the Laboratory Animal Science Committee of the IAPG CAS, Quadrants of mandibles were dissected, fixed in 4% buffered paraformaldehyde, decalcified in buffered EDTA (Sigma Aldrich, St. Louis, MO), dehydrated in ethanol series, treated with xylene and embedded in paraffin. Serial sagittal and frontal histological sections were processed and split between slides for the following analyses: haematoxylin–eosin (H&E) staining (morphology); BMP2; BMP6; BMP7; proliferating cell nuclear antigen (PCNA); osteocalcin (OCN); tartrate-resistant acid phosphatase (TRAP) and apoptotic DNA breaks (TUNEL). Immunohistochemistry After deparaffinization (xylen), rehydration (a gradient series of ethanols), antigen retrieval was applied to BMP6 sections (citrate buffer, pH ¼ 6.0/98 C/5 min/water bath). Endogenous
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peroxidase activity was eliminated in all sections by 3% hydrogen peroxide in phosphate-buffered saline (RT/5 min) and blocking serum was applied (RT/1 h) to eliminate possible background. The primary antibodies (anti-BMP6/ 2 mg/ml, Abcam (Cambridge, UK)/ab15640; anti-PCNA/2 mg/ ml, Santa Cruz Biotechnology (Santa Cruz, CA)/sc-7907 and anti-OCN/2.5 mg/ml, Abcam/ab93876) were applied overnight at 4 C. The primary antibodies (anti-BMP2/5 mg/ml, Abcam/ ab82511 and anti-BMP7/5 mg/ml, Abcam/ab56023) were applied for 1 h at RT. To visualize the primary antibody, a peroxidase-conjugated streptavidin-biotin system (Vectastain PK-4002; Vector Laboratories, Inc., Burlingame, CA) followed by chromogen substrate [diaminobenzidine (DAB); K3466; Dako, Copenhagen, Denmark] reaction were applied. Samples were counterstained with haematoxylin to clearly distinguish positive cells (in brown) from the negative cells (in blue). TRAP staining TRAP staining was used for detection of osteoclast activity. After deparaffinization and rehydration, slides were immersed in a reaction mixture prepared according to the manufacturer’s directions (387A-1KT; Sigma-Aldrich, St. Louis, MO) and kept at 37 C for 1.5 h to achieve the colour reaction with Fast Red substrate. Methyl green was used as a counterstain of Fast Red. TUNEL assay TdT-mediated dUTP-biotin nick end labelling (TUNEL, S7100; Chemicon-Millipore, Billerica, MA) was used to detect apoptotic DNA breaks. After deparaffinization and rehydration, elimination of endogenous peroxidase activity was achieved by 3% hydrogen peroxide in PBS (RT/5 min) and sections were pretreated with 20 mg/ml of proteinase K (Milipore) diluted in PBS (RT/15 min). Equilibration buffer (RT/15 min) was applied, followed by the working solution (37 C/45 min/humidified chamber). Finally, sections were incubated in anti-digoxigenin conjugate (RT/30 min) and the colour reaction was achieved by reaction of POD with DAB substrate. Samples were counterstained by haematoxylin to contrast the positive nuclei (in brown) against the background (in blue). 3D reconstruction Mouse heads (stages P14–P20) were fixed in Bouin’s solution and dehydrated in alcohol of increasing concentration, up to 100%. Depending on the size and gross preparation, the specimens were decalcified using EDTA for 2–30 days. Paraffin embedding was carried out according to standard procedures, and the specimens were cut as 7-mm thick serial sections in frontal planes. Routine staining was performed with H&E, OCN was immunohistochemically stained (EnVision System, Hamburg, Germany) for bone apposition and TRAP staining (Sigma, Deisenhofen, Germany) was used to identify bone resorption. The single sections were brought into alignment according to the general rules of 3D reconstructions (25).
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DOI: 10.3109/03008207.2014.951441
Distribution of BMP6 in the alveolar bone
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Figure 1. M1 at pre-eruption P15 stage. Distribution of BMP6, proliferation, OCN, osteoclast activity and apoptosis. H&E staining of the mandibular M1 at P15 (A and B) showing the root region and surrounding alveolar bone along the root (A1–5) and the tooth base (B1–5) in detail. BMP6 protein (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments and M1, first molar.
Results Distribution of BMP6 protein examined at pre-eruption stages At P15, the base of the bony socket was expanded so that the M1 was almost prepared for the eruption into the oral cavity. Secreted BMP6 protein was detected in mesenchymal cells in the freshly formed periodontium and in the layer of cementoblasts along the root, as well as in the osteoblasts lining the alveolus (Figure 1A1). Some BMP6-positive cells were also found at the base of the M1, around the apical part of the dental root (Figure 1B1). Proliferation was concentrated in mesenchymal cells of the periodontium around the root (Figure 1A2), where it positively correlated with BMP6 expression (Figure 1A1), and also in the most apical part of the Hertwig’s epithelial root sheath (HERS) (Figure 1B2). Few PCNA-positive cells were located at the growing alveolar bone in osteoblasts/osteocytes of the root region
(Figures 1A2 and B2). OCN localization along the inner margin of the alveolar crest beside the root positively correlated with BMP6 and PCNA (Figure 1A3). OCN was observed in root odontoblasts (Figure 1A3), as well as at the base of the newly created bone surrounding the tooth root (Figure 1B3). Osteoclast activity decreased at P15 stage: no active osteoclasts were found at the alveolar bone along the tooth root (Figure 1A4), and some scattered TRAP-positive osteoclasts were observed only in the alveolar bone beneath the M1 (Figure 1B4). Some dispersed cells of the periodontium (Figure 1A5) and the base of the tooth root at the alveolar bone (Figure 1B5) underwent apoptosis. A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptosis and TRAP-positive osteoclasts in the mandibular M1 at P15 can be seen in Figure 4(A). At the P22 stage, corresponding to the M1 at the P15 stage, the M3 was found to be prepared for eruption. BMP6-positive
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Figure 2. M3 at pre-eruption P22 stage. Distribution of BMP6, proliferation, OCN, osteoclast activity and apoptosis. H&E staining of the mandibular M3 at P22 (A and B) showing the root region and surrounding alveolar bone along the root (A1–5) and the tooth base. (B1–5). BMP6 (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments and M3, third molar.
cells were identified in the fibroblasts of the growing PDL and in cementoblasts along the root (Figure 2A1). BMP6 protein was also apparent at the base of the bony crypt, especially in the mesenchymal cells and osteoblasts at the tips of the alveolar bone (Figure 2B1). PCNA-positive fibroblasts were detected, together with PCNA-positive osteoblasts at the side of the alveolar bone (Figure 2A2). As BMP6-positive cells, also proliferating mesenchymal cells and osteoblasts were observed at the base of the bony crypt (Figure 2B2). The alveolar bone beside and under the tooth crypt was positive for OCN protein (Figure 2A3 and B3). Bone resorption was reduced to only some interspersed cells at the tooth base (Figure 2B4). No TRAP-positive cells were in the alveolar bone surrounding the tooth (Figure 2A4). Along with these findings, apoptotic cells were present only in the mesenchymal cells of the periodontium (Figure 2A5) and at the base of the bony crypt (Figure 2B5). A schematic illustration of the
spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive osteoclasts in the mandibular M3 at P22 can be seen in Figure 4(B). Detection of BMP6 protein at eruption stages At P19, the M1 had erupted in the oral cavity. BMP6-positive cells were observed among fibroblasts of the PDL along the M1 root, but mainly concentrated in the cells tightly contiguous with the dentine and inner surface of the alveolar bone (Figure 3A1). Cell proliferation accompanied BMP6 expression in fibroblasts of the PDL and in osteoblasts of the surrounding alveolar bone (Figure 3A2). OCN protein was present in the bone matrix (Figure 3A3). Osteoclast activity ceased and no TRAP-positive cells were observed in the surrounding bone (Figure 3A4). Apoptotic elimination was steadily decreased with just a few TUNEL-positive
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Figure 3. Comparison of eruption stages M1 (P19) and M3 (P26). Distribution of BMP6, proliferation, OCN, osteoclasts and apoptosis. H&E staining of the mandibular M1 at P19 (A) and M3 at P26 (B). The detailed pictures are focused on the inter-radicular root region of M1 (A1–5) and M3 (B1–5). BMP6 (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments; ir, inter-radicular region, M1, first molar; M2, second molar and M3, third molar.
osteoblasts/osteocytes in the alveolar bone (Figure 3A5). A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive osteoclasts in the mandibular M1 at P19 can be seen in Figure 4(C). To compare the same developmental stage of the M3, P26 was evaluated. BMP6-positive cells and proliferation activity still appeared in the periodontal region around the M3 root (Figure 3B1). Proliferating cells were concentrated at the base of the bony socket, near the tooth root and cementoblasts (Figure 3B2). Similarly to the corresponding stage of the M1, OCN-positive cells were observed in the alveolar bone and bone matrix (Figure 3B3). Bone turnover apparently slowed down, as no osteoclast activity (Figure 3B4) and no apoptotic cells were found in that region (Figure 3B5). A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive
osteoclasts in the mandibular M3 at P26 can be seen in Figure 4(D). Bone resorption and apposition around mouse molar eruption in 3D models To provide a more illustrative view of the bone resorption and apposition around the molars, 3D models were composed. At the P14 stage (Figure 5A1), resorption was predominantly in the surrounding alveolar bone of the M1 due to expansion of the roots (Figure 5B1). At that stage, the bone around the still small M3 dental primordium displayed apposition (Figure 5B1). At the P16 stage, the formation of the interdental septa was concomitant with higher bone apposition all over that area (Figure 5B2). The resorption had slowed down, whereas there were scattered regions of resorption around the M1 (Figure 5B2). At the P20 stage,
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Figure 4. Schematic illustration of the spatial distribution of BMP6, OCN, PCNA, TRAP-positive osteoclasts and apoptosis. Distribution is colour coded by spots: BMP6 (red spots), OCN (green spots), PCNA (brown spots), TRAP-positive osteoclasts (pink spots) and TUNEL-positive cells (blue spots) (Color picture is available online).
the growth of M3 was reflected by the prevalence of resorption within the crypt (Figure 5B3). Formation of the interdental septa continued as apposition, and resorption was visible at the lingual margin of the alveolar crest (Figure 5B3). Distribution of BMP2 and BMP7 proteins correlated with BMP6 expression pattern Pre-erupted stage of the M1 (P15) contained both BMP2 and BMP7-positive cells. The BMP2 as well as BMP7 proteins were apparent in osteoblasts/osteocytes at the top of the growing alveolar crest beside the root (Figure 6A1 and A2) and at the base of the newly formed alveolar bone surrounding the root sheath (Figure 6B1 and B2). Some positive cells were interspersed in periodontium (Figure 6A1 and A2). BMP2 and BMP7 proteins were observed also in odontoblasts (Figure 6A1 and A2). BMP2 and BMP7 were analysed also in the M3 at the stage P22 corresponding to the stage P15 (M1). BMP2 positivity was found in osteoblasts and osteocytes along the
alveolar bone surrounding the fibroblasts of the growing PDL (Figure 6C1). The expression of BMP7 overlapped with BMP2-positive cells (Figure 6C2). At the base of the bony crypt, especially along the root sheath, BMP2 protein was detected in the osteoblasts at the tips of the alveolar bone and in the osteoclasts (Figure 6D1). BMP7-positive cells were present in the same region (Figure 6D2). Moreover, also odontoblasts were BMP2 and BMP7 positive (Figure 6C1 and C2). At the stage P19, BMP2 and BMP7 expression accompanied BMP6-positive cells in osteoblasts at the inner surface of the alveolar bone and cells tightly contiguous with the dentine fibroblasts of the PDL along the M1 root (Figure 6E1 and E2). At P26, the corresponding developmental stage of M3, BMP2-positive cells were still detected in osteoblasts at the tips of the alveolar bone, occasionally in some osteocytes, and fibroblasts cells of the PDL (Figure 6F1). BMP7 expression pattern mostly overlapped with BMP2-positive cells. Alveolar bone surrounding the PDL was BMP7 positive as well as PDL fibroblasts (Figure 6F2).
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Figure 5. 3D reconstruction of the molar region. 3D reconstructions prepared from serial sections of the right half of the mandible with the molars and their surrounding bone in an anterior and 45 cranial view showing the M1, M2, M3, the ramus, the body and the alveolar bone of the corresponding part of the mandible (Figure A) at P14 (A1), P16 (A2) and P20 (A3). Enamel is displayed in blue (lost in P16 for M1 and in P20 for M1 and for M2 due to histological processing). The mandibular canal with the inferior alveolar nerve is visible (yellow) at the foremost section plane. The row of Figure B shows the corresponding reconstructions, with the teeth removed and the alveolar bone exposed. Regions of bone apposition are colour coded with red and regions of bone resorption are coded with green. Scale bar ¼ 1000 mm. IDS, interdental space; IRS, inter-radicular space; M1, first molar; M2, second molar and M3, third molar (Color picture is available online).
Discussion The most dynamic changes in the alveolar bone occur together with root development and tooth eruption (26). Our findings showed that BMP6 protein was present in the alveolar bone, mainly along the base of the tooth crypt at the pre-eruption stage of the M1 (at P15). Furthermore, BMP6-positive cells were observed around the apical part of the dental root. Bone formation at the tooth base was confirmed by detection of PCNA-positive cells and increasing expression of the late osteoblastic marker OCN (27). Colocalization of BMP6, OCN and other BMPs proteins: BMP2 and BMP7, suggested a dynamic bone formation around eruption. BMP2 and BMP7 were observed coexpressed in a number of tissues that are known to be the source of inductive signals, and promote intense growth during skeletogenesis (28,29). Moreover, a number of apoptotic cells and active osteoclasts scattered at the
base of the bony crypt confirmed low osteoclastogenesis. This alveolar bone formation at the tooth base is important for eruption, especially during its intraosseous phase (4), when the tooth moves to its final occlusal plane. Several BMPs participate in alveolar bone formation at the base of the bony crypt, but BMP6 seems to be essential for proper development as shown also experimentally (20). Inhibition of Bmp6 using local injection of siRNA caused inhibited alveolar bone formation, reduction in the amount of alveolar bone in the crypt at the base of the impacted first molar, in comparison with adjacent second molar that erupted. In many instances, the alveolar bone in the bony crypt at the base of the erupted molar also appeared more compact than the comparable alveolar bone of the impacted molar (20). The bone regression process occurs earlier, to provide time to remodel the alveolar bone surrounding mouse mandibular molars (3). The M1 at the P10 stage and M3 at the P17 stage
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Figure 6. Distribution of BMP2 and BMP7 at pre-erupted and erupted stages of M1 and M3. BMP6 staining of the mandibular M1 at stage P15 (A and B), M3 at stage P22 (C and D) at pre-erupted stages, M1 at stage P19 (E) and M3 at stage P26 (F) at erupted stages, showing the root region and surrounding alveolar bone along the root and the tooth base. BMP2 (A1, B1, C1, D1, E1 and F1) and BMP7 (A2, B2, C2, D2, E2 and F2). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments; ir, inter-radicular region; M1, first molar; M2, second molar and M3, third molar.
Distribution of BMP6 in the alveolar bone
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start to show significant root elongation (24,30) and the simultaneously increasing activity of osteoclasts is likely to create the space for proper PDL development. This indicates another role for BMP6 in stimulating the generation of osteoclasts. A stimulatory effect of BMPs on osteoclasts was reported earlier (31). Moreover, mature osteoclasts can also synthesize BMPs (32), including BMP6 protein (33). The expression of BMP6 in active osteoclasts at resorption sites seems to indicate its involvement in the intercellular communication loop that leads to osteoblastic recruitment and differentiation, as reported in other studies (32,34). BMP2 alone does not induce osteoclasts formation (35); nevertheless, BMP2/4 is a prerequisite for osteoclastogenesis. Taken together, BMP6 and other BMPs might be very important regulators of bone homeostasis. In general, osteoclastogenesis dominated prior to eruption, during expansion of the roots and culminated around the P14 in the M1 and the P21 in the M3. Later, osteoclast activity decreased and the alveolar bone displayed prevalent apposition. BMP6 expression positively correlated with proliferation and bone formation. This is consistent with the regulatory role of BMPs in cell proliferation, differentiation and apoptosis in embryonic and postnatal osteoblasts and osteoclasts (36). Additionally, BMP6 protein was persistent in the alveolar bone up to eruption. The mouse M1 erupts by P20 (30), M2 eruption occurs 1 day later and eruption of the M3 1 week later (24). The growth of tooth germs is constrained by the surrounding tissues (37); however, during eruption, the different bone environment seemed to have no effect on distribution of BMP6 protein in the alveolar bone around the M1 and M3 at the same stage of odontogenesis. Fully erupted M1/M3 showed fewer BMP6positive cells, but BMP6 expression was persistent in the surrounding periodontal tissue. Furthermore, BMP2 and BMP7 proteins were detected in the alveolar bone surrounding the tooth and periodontal ligaments. BMP7-positive cells of the forming periodontal ligament were immunopositive as well as in the layer of fully functional odontoblasts (17). This is in agreement with in vitro data showing odontogenic and osteogenic differentiation of hTGSCs promoted by BMP2 and BMP7 (38). Our results further showed localization of BMP6 protein in the developing periodontium, where the timing corresponded with the rapid growth of PDL and cementum around the M1 (30). These structures develop along with formation of the roots during tooth eruption. The growth of PDL was confirmed by detection of proliferation in that region and lower numbers of apoptotic cells. PDL is required for eruption, as demonstrated by surgical studies in the dog (39). Moreover, Wise et al. (40) confirmed the important role of the dental follicle in the molecular control of osteoclastogenesis. The signalling and interactions among dental follicle cells, osteoblasts and osteoclasts are involved in the complex interplay of regulatory events during tooth eruption. BMP6 protein apparently contributes to interactions among these structures.
Acknowledgments The authors would like to thank Ms. Lucie Vrlikova´ for her valuable technical assistance, Mrs. B. Danielowski, Mrs. I.
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Schwarz for their skilful processing of the histological material and for creating the 3D-reconstructions from the serial sections, and Mr. R. Mey for the reconstructions of P16.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a joint project of the Grant Agency of the Czech Republic (P302/12/J059) and the Deutsche Forschungsgemeinschaft (Ra428/1-11). The IAPG labs run under the RVO 67985904.
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ORIGINAL RESEARCH
Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption Veronika Oralova´1,2, Ivana Chlasta´kova´3, Ralf Johannes Radlanski4, and Eva Matalova´1,3
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1
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Brno, Czech Republic, 2Department of Experimental Biology, Masaryk University, Brno, Czech Republic, 3Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic, and 4Department of Craniofacial Developmental Biology, Charite´-Universita¨tsmedizin, Berlin, Germany
Abstract
Keywords
Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth– bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.
Bone morphogenetic protein, bone resorption, bone apposition, periodontal ligament, three-dimensional images
Introduction The tooth is a typical example of an organ formed by epithelial–mesenchymal interactions (1). Different morphogens [bone morphogenetic protein (BMPs), WNTs, FGFs and HHs] participate in odontogenesis and related osteogenesis. Proper dentition is formed by functional reciprocal interplay of the tooth and surrounding structures (2,3). Tooth eruption is a highly regulated multifactorial process involving formation and reorganization of tissues coordinated by a complex set of molecular events. According to previous studies, biological tooth movement in mice is a result of osteoclastic bone resorption combined with alveolar bone and cementum formation (4,5). Prior to eruption (around postnatal stage 10), mononuclear cells move to the dental follicle and fuse to form osteoclasts, which resorb alveolar bone to create the eruption pathway (6). In addition, remodelling of the collagenous Correspondence: Ms Veronika Oralova´, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Veveri 97, Brno 602 00, Czech Republic. Tel: +420-532-290-163. Fax: +420-541212-988. E-mail: [email protected]
History Received 1 April 2014 Revised 23 July 2014 Accepted 26 July 2014 Published online 26 August 2014
extracellular matrix, periodontal ligaments (PDL) and alveolar bone was identified as a key molecular process facilitating tooth movement (7,8). Bone morphogenetic proteins (BMPs) are secretory signalling molecules belonging to the transforming growth factor-b (TGF-b) superfamily of polypeptides. Along with the capability to induce ectopic bone formation, they have other important signalling functions, such as governing cell proliferation, differentiation and apoptosis (9). BMPs were described to be expressed at different stages throughout tooth development and have central roles in tooth initiation and morphogenesis (10). BMP2 and BMP4 were shown to mimic some of the functions of the dental epithelium and induce tooth germ initiation in the mesenchyme (11). Moreover, BMP2 and BMP4 are also involved in osteoinduction (12,13). BMP2 and BMP7 stimulate mouse mesenchymal cells to differentiate into osteoblasts (14). BMP2 secreted in the basal region of the mouse dental follicle (DF) commits these cells towards the cementoblast/osteoblast phenotype (15) and supports the expression of cementum attachment protein (CA) in human periodontal ligament cells (16).
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BMP2, -3 and -7 were reported in the pre-dentine, dentine, osteoblasts, osteocytes, osteoid, chondrocytes, alveolar bone, cementum, PDL and other craniofacial structures (17,18). BMP6 was shown to function at early and late stages of osteogenic differentiation in human dental follicle cells (12). BMP6 was found to be upregulated in human PDL, suggesting the role of this protein in tooth eruption (19,20). BMP6 expression was found in cultured pulp cells and, together with other BMPs, BMP6 appears to be potent protein for future treatment strategies of bone regeneration (21). BMP6 knockout mice are viable, no dental defects have been reported, and the only skeletal alterations published so far are delayed ossification of the sternum (22) and decreased bone volume/ trabecular volume in the long bones (23). Knowledge about BMP6 in bones comes mostly from endochondral bones; nevertheless, local injection of a small-interfering RNA (siRNA) targeting BMP6 inhibited alveolar bone formation and subsequent tooth eruption, despite the fact that the eruption pathway was formed (20). The aim of this study was to further contribute to knowledge about BMP6 in relation to tooth eruption by analysis of its temporospatial expression pattern in mandibular molar teeth and surrounding structures, particularly the intramembranous bone, during pre-eruption and eruption stages of the first mouse molar tooth (M1). Attention was paid to localization of BMP6 and its correlation with proliferation, differentiation and apoptosis, as well as bone apposition/ resorption. Additionally, the pattern of BMP6 protein was matched with localization of other BMPs (BMP2 and BMP7). With respect to different bone environments during first/ second and third molar eruptions (24), distribution of BMP6 protein was compared in the bone around the first and third mouse molars. Moreover, an overview of tooth–bone development was documented using three-dimensional models (3D).
Materials and methods Animals Wild-type mice were obtained from the Breeding Unit of the Masaryk University in Brno. Heads of pups corresponding to postnatal (P) days up to the P26 were collected for sampling. All procedures were carried out according to the experimental protocol approved by the Laboratory Animal Science Committee of the IAPG CAS, Quadrants of mandibles were dissected, fixed in 4% buffered paraformaldehyde, decalcified in buffered EDTA (Sigma Aldrich, St. Louis, MO), dehydrated in ethanol series, treated with xylene and embedded in paraffin. Serial sagittal and frontal histological sections were processed and split between slides for the following analyses: haematoxylin–eosin (H&E) staining (morphology); BMP2; BMP6; BMP7; proliferating cell nuclear antigen (PCNA); osteocalcin (OCN); tartrate-resistant acid phosphatase (TRAP) and apoptotic DNA breaks (TUNEL). Immunohistochemistry After deparaffinization (xylen), rehydration (a gradient series of ethanols), antigen retrieval was applied to BMP6 sections (citrate buffer, pH ¼ 6.0/98 C/5 min/water bath). Endogenous
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peroxidase activity was eliminated in all sections by 3% hydrogen peroxide in phosphate-buffered saline (RT/5 min) and blocking serum was applied (RT/1 h) to eliminate possible background. The primary antibodies (anti-BMP6/ 2 mg/ml, Abcam (Cambridge, UK)/ab15640; anti-PCNA/2 mg/ ml, Santa Cruz Biotechnology (Santa Cruz, CA)/sc-7907 and anti-OCN/2.5 mg/ml, Abcam/ab93876) were applied overnight at 4 C. The primary antibodies (anti-BMP2/5 mg/ml, Abcam/ ab82511 and anti-BMP7/5 mg/ml, Abcam/ab56023) were applied for 1 h at RT. To visualize the primary antibody, a peroxidase-conjugated streptavidin-biotin system (Vectastain PK-4002; Vector Laboratories, Inc., Burlingame, CA) followed by chromogen substrate [diaminobenzidine (DAB); K3466; Dako, Copenhagen, Denmark] reaction were applied. Samples were counterstained with haematoxylin to clearly distinguish positive cells (in brown) from the negative cells (in blue). TRAP staining TRAP staining was used for detection of osteoclast activity. After deparaffinization and rehydration, slides were immersed in a reaction mixture prepared according to the manufacturer’s directions (387A-1KT; Sigma-Aldrich, St. Louis, MO) and kept at 37 C for 1.5 h to achieve the colour reaction with Fast Red substrate. Methyl green was used as a counterstain of Fast Red. TUNEL assay TdT-mediated dUTP-biotin nick end labelling (TUNEL, S7100; Chemicon-Millipore, Billerica, MA) was used to detect apoptotic DNA breaks. After deparaffinization and rehydration, elimination of endogenous peroxidase activity was achieved by 3% hydrogen peroxide in PBS (RT/5 min) and sections were pretreated with 20 mg/ml of proteinase K (Milipore) diluted in PBS (RT/15 min). Equilibration buffer (RT/15 min) was applied, followed by the working solution (37 C/45 min/humidified chamber). Finally, sections were incubated in anti-digoxigenin conjugate (RT/30 min) and the colour reaction was achieved by reaction of POD with DAB substrate. Samples were counterstained by haematoxylin to contrast the positive nuclei (in brown) against the background (in blue). 3D reconstruction Mouse heads (stages P14–P20) were fixed in Bouin’s solution and dehydrated in alcohol of increasing concentration, up to 100%. Depending on the size and gross preparation, the specimens were decalcified using EDTA for 2–30 days. Paraffin embedding was carried out according to standard procedures, and the specimens were cut as 7-mm thick serial sections in frontal planes. Routine staining was performed with H&E, OCN was immunohistochemically stained (EnVision System, Hamburg, Germany) for bone apposition and TRAP staining (Sigma, Deisenhofen, Germany) was used to identify bone resorption. The single sections were brought into alignment according to the general rules of 3D reconstructions (25).
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Figure 1. M1 at pre-eruption P15 stage. Distribution of BMP6, proliferation, OCN, osteoclast activity and apoptosis. H&E staining of the mandibular M1 at P15 (A and B) showing the root region and surrounding alveolar bone along the root (A1–5) and the tooth base (B1–5) in detail. BMP6 protein (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments and M1, first molar.
Results Distribution of BMP6 protein examined at pre-eruption stages At P15, the base of the bony socket was expanded so that the M1 was almost prepared for the eruption into the oral cavity. Secreted BMP6 protein was detected in mesenchymal cells in the freshly formed periodontium and in the layer of cementoblasts along the root, as well as in the osteoblasts lining the alveolus (Figure 1A1). Some BMP6-positive cells were also found at the base of the M1, around the apical part of the dental root (Figure 1B1). Proliferation was concentrated in mesenchymal cells of the periodontium around the root (Figure 1A2), where it positively correlated with BMP6 expression (Figure 1A1), and also in the most apical part of the Hertwig’s epithelial root sheath (HERS) (Figure 1B2). Few PCNA-positive cells were located at the growing alveolar bone in osteoblasts/osteocytes of the root region
(Figures 1A2 and B2). OCN localization along the inner margin of the alveolar crest beside the root positively correlated with BMP6 and PCNA (Figure 1A3). OCN was observed in root odontoblasts (Figure 1A3), as well as at the base of the newly created bone surrounding the tooth root (Figure 1B3). Osteoclast activity decreased at P15 stage: no active osteoclasts were found at the alveolar bone along the tooth root (Figure 1A4), and some scattered TRAP-positive osteoclasts were observed only in the alveolar bone beneath the M1 (Figure 1B4). Some dispersed cells of the periodontium (Figure 1A5) and the base of the tooth root at the alveolar bone (Figure 1B5) underwent apoptosis. A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptosis and TRAP-positive osteoclasts in the mandibular M1 at P15 can be seen in Figure 4(A). At the P22 stage, corresponding to the M1 at the P15 stage, the M3 was found to be prepared for eruption. BMP6-positive
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Figure 2. M3 at pre-eruption P22 stage. Distribution of BMP6, proliferation, OCN, osteoclast activity and apoptosis. H&E staining of the mandibular M3 at P22 (A and B) showing the root region and surrounding alveolar bone along the root (A1–5) and the tooth base. (B1–5). BMP6 (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments and M3, third molar.
cells were identified in the fibroblasts of the growing PDL and in cementoblasts along the root (Figure 2A1). BMP6 protein was also apparent at the base of the bony crypt, especially in the mesenchymal cells and osteoblasts at the tips of the alveolar bone (Figure 2B1). PCNA-positive fibroblasts were detected, together with PCNA-positive osteoblasts at the side of the alveolar bone (Figure 2A2). As BMP6-positive cells, also proliferating mesenchymal cells and osteoblasts were observed at the base of the bony crypt (Figure 2B2). The alveolar bone beside and under the tooth crypt was positive for OCN protein (Figure 2A3 and B3). Bone resorption was reduced to only some interspersed cells at the tooth base (Figure 2B4). No TRAP-positive cells were in the alveolar bone surrounding the tooth (Figure 2A4). Along with these findings, apoptotic cells were present only in the mesenchymal cells of the periodontium (Figure 2A5) and at the base of the bony crypt (Figure 2B5). A schematic illustration of the
spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive osteoclasts in the mandibular M3 at P22 can be seen in Figure 4(B). Detection of BMP6 protein at eruption stages At P19, the M1 had erupted in the oral cavity. BMP6-positive cells were observed among fibroblasts of the PDL along the M1 root, but mainly concentrated in the cells tightly contiguous with the dentine and inner surface of the alveolar bone (Figure 3A1). Cell proliferation accompanied BMP6 expression in fibroblasts of the PDL and in osteoblasts of the surrounding alveolar bone (Figure 3A2). OCN protein was present in the bone matrix (Figure 3A3). Osteoclast activity ceased and no TRAP-positive cells were observed in the surrounding bone (Figure 3A4). Apoptotic elimination was steadily decreased with just a few TUNEL-positive
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Figure 3. Comparison of eruption stages M1 (P19) and M3 (P26). Distribution of BMP6, proliferation, OCN, osteoclasts and apoptosis. H&E staining of the mandibular M1 at P19 (A) and M3 at P26 (B). The detailed pictures are focused on the inter-radicular root region of M1 (A1–5) and M3 (B1–5). BMP6 (A1 and B1), PCNA (A2 and B2), OCN (A3 and B3), TRAP staining (A4 and B4) and apoptosis (A5 and B5). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments; ir, inter-radicular region, M1, first molar; M2, second molar and M3, third molar.
osteoblasts/osteocytes in the alveolar bone (Figure 3A5). A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive osteoclasts in the mandibular M1 at P19 can be seen in Figure 4(C). To compare the same developmental stage of the M3, P26 was evaluated. BMP6-positive cells and proliferation activity still appeared in the periodontal region around the M3 root (Figure 3B1). Proliferating cells were concentrated at the base of the bony socket, near the tooth root and cementoblasts (Figure 3B2). Similarly to the corresponding stage of the M1, OCN-positive cells were observed in the alveolar bone and bone matrix (Figure 3B3). Bone turnover apparently slowed down, as no osteoclast activity (Figure 3B4) and no apoptotic cells were found in that region (Figure 3B5). A schematic illustration of the spatial distribution of BMP6, OCN, PCNA, apoptotic cells and TRAP-positive
osteoclasts in the mandibular M3 at P26 can be seen in Figure 4(D). Bone resorption and apposition around mouse molar eruption in 3D models To provide a more illustrative view of the bone resorption and apposition around the molars, 3D models were composed. At the P14 stage (Figure 5A1), resorption was predominantly in the surrounding alveolar bone of the M1 due to expansion of the roots (Figure 5B1). At that stage, the bone around the still small M3 dental primordium displayed apposition (Figure 5B1). At the P16 stage, the formation of the interdental septa was concomitant with higher bone apposition all over that area (Figure 5B2). The resorption had slowed down, whereas there were scattered regions of resorption around the M1 (Figure 5B2). At the P20 stage,
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Figure 4. Schematic illustration of the spatial distribution of BMP6, OCN, PCNA, TRAP-positive osteoclasts and apoptosis. Distribution is colour coded by spots: BMP6 (red spots), OCN (green spots), PCNA (brown spots), TRAP-positive osteoclasts (pink spots) and TUNEL-positive cells (blue spots) (Color picture is available online).
the growth of M3 was reflected by the prevalence of resorption within the crypt (Figure 5B3). Formation of the interdental septa continued as apposition, and resorption was visible at the lingual margin of the alveolar crest (Figure 5B3). Distribution of BMP2 and BMP7 proteins correlated with BMP6 expression pattern Pre-erupted stage of the M1 (P15) contained both BMP2 and BMP7-positive cells. The BMP2 as well as BMP7 proteins were apparent in osteoblasts/osteocytes at the top of the growing alveolar crest beside the root (Figure 6A1 and A2) and at the base of the newly formed alveolar bone surrounding the root sheath (Figure 6B1 and B2). Some positive cells were interspersed in periodontium (Figure 6A1 and A2). BMP2 and BMP7 proteins were observed also in odontoblasts (Figure 6A1 and A2). BMP2 and BMP7 were analysed also in the M3 at the stage P22 corresponding to the stage P15 (M1). BMP2 positivity was found in osteoblasts and osteocytes along the
alveolar bone surrounding the fibroblasts of the growing PDL (Figure 6C1). The expression of BMP7 overlapped with BMP2-positive cells (Figure 6C2). At the base of the bony crypt, especially along the root sheath, BMP2 protein was detected in the osteoblasts at the tips of the alveolar bone and in the osteoclasts (Figure 6D1). BMP7-positive cells were present in the same region (Figure 6D2). Moreover, also odontoblasts were BMP2 and BMP7 positive (Figure 6C1 and C2). At the stage P19, BMP2 and BMP7 expression accompanied BMP6-positive cells in osteoblasts at the inner surface of the alveolar bone and cells tightly contiguous with the dentine fibroblasts of the PDL along the M1 root (Figure 6E1 and E2). At P26, the corresponding developmental stage of M3, BMP2-positive cells were still detected in osteoblasts at the tips of the alveolar bone, occasionally in some osteocytes, and fibroblasts cells of the PDL (Figure 6F1). BMP7 expression pattern mostly overlapped with BMP2-positive cells. Alveolar bone surrounding the PDL was BMP7 positive as well as PDL fibroblasts (Figure 6F2).
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Figure 5. 3D reconstruction of the molar region. 3D reconstructions prepared from serial sections of the right half of the mandible with the molars and their surrounding bone in an anterior and 45 cranial view showing the M1, M2, M3, the ramus, the body and the alveolar bone of the corresponding part of the mandible (Figure A) at P14 (A1), P16 (A2) and P20 (A3). Enamel is displayed in blue (lost in P16 for M1 and in P20 for M1 and for M2 due to histological processing). The mandibular canal with the inferior alveolar nerve is visible (yellow) at the foremost section plane. The row of Figure B shows the corresponding reconstructions, with the teeth removed and the alveolar bone exposed. Regions of bone apposition are colour coded with red and regions of bone resorption are coded with green. Scale bar ¼ 1000 mm. IDS, interdental space; IRS, inter-radicular space; M1, first molar; M2, second molar and M3, third molar (Color picture is available online).
Discussion The most dynamic changes in the alveolar bone occur together with root development and tooth eruption (26). Our findings showed that BMP6 protein was present in the alveolar bone, mainly along the base of the tooth crypt at the pre-eruption stage of the M1 (at P15). Furthermore, BMP6-positive cells were observed around the apical part of the dental root. Bone formation at the tooth base was confirmed by detection of PCNA-positive cells and increasing expression of the late osteoblastic marker OCN (27). Colocalization of BMP6, OCN and other BMPs proteins: BMP2 and BMP7, suggested a dynamic bone formation around eruption. BMP2 and BMP7 were observed coexpressed in a number of tissues that are known to be the source of inductive signals, and promote intense growth during skeletogenesis (28,29). Moreover, a number of apoptotic cells and active osteoclasts scattered at the
base of the bony crypt confirmed low osteoclastogenesis. This alveolar bone formation at the tooth base is important for eruption, especially during its intraosseous phase (4), when the tooth moves to its final occlusal plane. Several BMPs participate in alveolar bone formation at the base of the bony crypt, but BMP6 seems to be essential for proper development as shown also experimentally (20). Inhibition of Bmp6 using local injection of siRNA caused inhibited alveolar bone formation, reduction in the amount of alveolar bone in the crypt at the base of the impacted first molar, in comparison with adjacent second molar that erupted. In many instances, the alveolar bone in the bony crypt at the base of the erupted molar also appeared more compact than the comparable alveolar bone of the impacted molar (20). The bone regression process occurs earlier, to provide time to remodel the alveolar bone surrounding mouse mandibular molars (3). The M1 at the P10 stage and M3 at the P17 stage
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Figure 6. Distribution of BMP2 and BMP7 at pre-erupted and erupted stages of M1 and M3. BMP6 staining of the mandibular M1 at stage P15 (A and B), M3 at stage P22 (C and D) at pre-erupted stages, M1 at stage P19 (E) and M3 at stage P26 (F) at erupted stages, showing the root region and surrounding alveolar bone along the root and the tooth base. BMP2 (A1, B1, C1, D1, E1 and F1) and BMP7 (A2, B2, C2, D2, E2 and F2). Black arrows point to positive cells in brown. Scale bar ¼ 100 mm. ab, alveolar bone; dp, dental pulp; pdl, periodontal ligaments; ir, inter-radicular region; M1, first molar; M2, second molar and M3, third molar.
Distribution of BMP6 in the alveolar bone
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start to show significant root elongation (24,30) and the simultaneously increasing activity of osteoclasts is likely to create the space for proper PDL development. This indicates another role for BMP6 in stimulating the generation of osteoclasts. A stimulatory effect of BMPs on osteoclasts was reported earlier (31). Moreover, mature osteoclasts can also synthesize BMPs (32), including BMP6 protein (33). The expression of BMP6 in active osteoclasts at resorption sites seems to indicate its involvement in the intercellular communication loop that leads to osteoblastic recruitment and differentiation, as reported in other studies (32,34). BMP2 alone does not induce osteoclasts formation (35); nevertheless, BMP2/4 is a prerequisite for osteoclastogenesis. Taken together, BMP6 and other BMPs might be very important regulators of bone homeostasis. In general, osteoclastogenesis dominated prior to eruption, during expansion of the roots and culminated around the P14 in the M1 and the P21 in the M3. Later, osteoclast activity decreased and the alveolar bone displayed prevalent apposition. BMP6 expression positively correlated with proliferation and bone formation. This is consistent with the regulatory role of BMPs in cell proliferation, differentiation and apoptosis in embryonic and postnatal osteoblasts and osteoclasts (36). Additionally, BMP6 protein was persistent in the alveolar bone up to eruption. The mouse M1 erupts by P20 (30), M2 eruption occurs 1 day later and eruption of the M3 1 week later (24). The growth of tooth germs is constrained by the surrounding tissues (37); however, during eruption, the different bone environment seemed to have no effect on distribution of BMP6 protein in the alveolar bone around the M1 and M3 at the same stage of odontogenesis. Fully erupted M1/M3 showed fewer BMP6positive cells, but BMP6 expression was persistent in the surrounding periodontal tissue. Furthermore, BMP2 and BMP7 proteins were detected in the alveolar bone surrounding the tooth and periodontal ligaments. BMP7-positive cells of the forming periodontal ligament were immunopositive as well as in the layer of fully functional odontoblasts (17). This is in agreement with in vitro data showing odontogenic and osteogenic differentiation of hTGSCs promoted by BMP2 and BMP7 (38). Our results further showed localization of BMP6 protein in the developing periodontium, where the timing corresponded with the rapid growth of PDL and cementum around the M1 (30). These structures develop along with formation of the roots during tooth eruption. The growth of PDL was confirmed by detection of proliferation in that region and lower numbers of apoptotic cells. PDL is required for eruption, as demonstrated by surgical studies in the dog (39). Moreover, Wise et al. (40) confirmed the important role of the dental follicle in the molecular control of osteoclastogenesis. The signalling and interactions among dental follicle cells, osteoblasts and osteoclasts are involved in the complex interplay of regulatory events during tooth eruption. BMP6 protein apparently contributes to interactions among these structures.
Acknowledgments The authors would like to thank Ms. Lucie Vrlikova´ for her valuable technical assistance, Mrs. B. Danielowski, Mrs. I.
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Schwarz for their skilful processing of the histological material and for creating the 3D-reconstructions from the serial sections, and Mr. R. Mey for the reconstructions of P16.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a joint project of the Grant Agency of the Czech Republic (P302/12/J059) and the Deutsche Forschungsgemeinschaft (Ra428/1-11). The IAPG labs run under the RVO 67985904.
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